The present series of experiments tested our hypothesis that the growth of a tumor, after radiation doses similar to those given in radiotherapy, has to rely on the vasculogenesis pathway, which is dependent on circulating cells, primarily of BM origin (Figure ), and that inhibition of this pathway will prevent, or delay, tumor recurrence. Our results in the i.c. implanted GBM in mice support this hypothesis. We demonstrate that there is a large influx of BMDCs in the irradiated tumors as they begin to regrow after irradiation, and that 3 different ways of abrogating this influx (inhibition of HIF-1 or SDF-1/CXCR4 or depletion of monocytes/macrophages) prevent, or substantially delay, tumor recurrence. Our data suggest that the stimulus for the influx of BMDCs is increased tumor hypoxia caused by loss of functional vasculature after irradiation. Notably, we demonstrate that hypoxia is dramatically increased in the well-vascularized U251 brain tumor as it initiates regrowth after irradiation and this coincides with increased tumor HIF-1 levels. Further, inhibition of HIF-1 or inhibition of the interaction of the HIF-1–upregulated SDF-1 with its receptor, CXCR4, after irradiation prevents both the recruitment of BMDCs into the tumors and their regrowth after irradiation.
Model of the main contributions of BMDCs; and cytokines that promote restoration of tumor vasculature after irradiation.
As discussed earlier, the 2 principal ways in which a tumor can expand its vasculature as it grows is by angiogenesis involving sprouting of endothelial cells from nearby normal vessels or by vasculogenesis, which occurs by the recruitment into the tumor of circulating endothelial and other cells. Our data suggest that for the intracranial growth of the U251 tumor, the angiogenesis pathway is dominant, as none of the 3 methods we used to inhibit vasculogenesis affected the growth of the unirradiated control tumors (Figures , 4, and 5). However, once the tumor received irradiation, most of the CD31+
endothelial cells in the tumor were eradicated (Supplemental Figure 2E). Also, those in the surrounding normal vessels would be expected to be sterilized, thereby inhibiting the angiogenesis pathway. Indeed, we and others have shown that irradiation abrogates tumor angiogenesis (45
). This loss of functional vasculature in the tumor combined with an inability of the normal vasculature to provide vessels is likely the reason for the large increase in tumor hypoxia we observed 2 weeks after irradiation (Figure , A and B) and the resulting increase in tumor HIF-1 levels (Figure C), thereby providing the stimulus for vasculogenesis. It has been reported that local irradiation promotes HIF-1 activation due to tumor reoxygenation and increased free radicals (28
). In our i.c. tumor model, HIF-1 was activated by local irradiation, but not by the same mechanism as reported, because it occurred much later than that caused by free radical production and was more consistent with the loss of tumor vasculature. Consistent with HIF-1 being the signal for vasculogenesis, we show that both pharmacological and genetic inhibition of HIF-1 attenuated BMDC recruitment and inhibited tumor recurrence. Of note is the fact that the effect of the HIF-1 inhibitor NSC-134754 is greater than that of HIF-1 KD or KO in the tumor cells (Figure and Supplemental Figure 3). The likely reason for this is that in the HIF-1–KO tumors there are also stromal cells that can express HIF-1 (24
AMD3100, an FDA-approved orphan drug, promotes the mobilization of BMDCs from the BM niche to peripheral blood by inhibiting the interaction of SDF-1 with CXCR4 (46
). As SDF-1 is also known to have an important role in retention of CXCR4+
BMDCs in perivascular hypoxic areas of ischemic tissue (7
), we used AMD3100 to inhibit this part of the vasculogenesis pathway. Although it has been reported that SDF-1 inhibition by AMD3100 increases the therapy-induced growth delay of GBM in a mouse model by inhibiting the Erk and Akt pathways (47
), this is not the mechanism of the effect we observed for 2 reasons. First, we only applied the drug after tumor irradiation, thus making any effect on tumor cell radiosensitivity unlikely. Second, we saw no effect of AMD3100 on the growth of the cells in vitro with or without irradiation. However, the effect of this drug in blocking BMDC influx and tumor regrowth was dramatic after either single or fractionated doses of irradiation. An important feature of our data is that although we completed the drug infusion before the tumors were eradicated, the tumors continued to shrink and did not recur even long after the end of the infusion.
The BMDC accumulation in the U251 tumors following irradiation was composed largely of CD11b+
monocytes. These cells have been shown to be increased in the BM, spleens, and peripheral blood of tumor-bearing mice and in the peripheral blood of cancer patients (48
). There is strong clinical evidence that infiltration levels of macrophages that are of myeloid cell lineage correlate with poor prognosis in breast, prostate, ovarian, and cervical cancers (50
). In our tumor model, irradiation significantly increased the number of CD11b+
myeloid cells in the tumor. These cells are highly proangiogenic, as we and others have reported, suggesting that they are attractive targets for enhancing tumor response to irradiation (20
). In addition, it has been reported that CD11b+
myeloid cells mediate tumor refractoriness to anti-VEGF treatment (51
). A significant finding in the irradiated tumors was that approximately 50% of the CD11b+
infiltrating monocytes also expressed the angiopoietin receptor Tie2, defining them as Tie2-expressing monocytes (TEMs). These have been shown to be highly related to tumor-associated macrophages (TAMs) and to express a gene signature associated with active remodeling and angiogenesis (52
). Significantly, these cells were undetectable in the unirradiated U251 tumors. Although it is possible that CD11b–
BMDCs contribute to the vasculogenesis after irradiation, we found no evidence for the involvement of EPCs. However, in other studies, we have found that antibody neutralization of CD11b had a significant radiosensitization effect on transplanted tumors, whereas depletion of Gr-1+
cells did not show the effect (G.O. Ahn et al., unpublished observations). This implicates CD11b+
cells as the important proangiogenic population, a conclusion similar to that of F. Pucci et al. (52
Anti-VEGF treatment combined with irradiation is an emerging strategy currently being tested in the treatment of GBM. However, recent reports of preclinical data indicate that the benefits are transitory, followed by rapid regrowth and increased tumor aggressiveness (53
). Our data also show that inhibiting the VEGF pathway through blockage of VEGFR2 by DC101 produces an increase in growth delay by irradiation. However, the effect was transitory, and the tumors regrew following the cessation of the treatment (Figure E). In addition, VEGF blockade was less effective than AMD3100 treatment in inhibiting tumor perfusion after irradiation (Figure F). Thus, blocking angiogenesis is inferior to inhibiting the vasculogenesis pathway in preventing both the postirradiation return of tumor perfusion and tumor recurrences.
In summary, our data suggest that the novel strategy of inhibiting the vasculogenesis pathway after local tumor irradiation has the potential to improve the control of human GBM by radiotherapy. In essence, this strategy abolishes the need to kill all the tumor cells, replacing it with the need only to kill the endothelial cells in and around the tumor, which will require significantly lower radiation doses than those needed to sterilize all the tumor cells.